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Phosphorylation of the microtubule-associated protein tau may be a good thing because it can speed up microtubule transport (see ARF related news story), but abnormal phosphorylation causes the protein to form paired-helical fragments, leading to the intracellular neurofibrillary tangles that are found in neurons in Alzheimer disease brains. What causes accumulation of abnormally phosphorylated tau? Have kinases run amok? Are proteases and phosphatases not doing their job? To answer these questions, we need to know more about these regulatory proteins. Three papers currently in press in the Journal of Biological Chemistry shed light on phosphatase and protease contributions to tau regulation.

Pat McGeer and colleagues at the University of British Columbia, Vancouver, report that tau is degraded by the protease thrombin. First author Testuaki Arai and colleagues incubated protease inhibitors with cytosolic extracts from human brain to see which inhibitors prevented proteolytic degradation of tau. (This work was also presented at the 34th annual meeting of the Society for Neuroscience, see SfN abstract 788.2.) When Arai used the thrombin-specific inhibitor PPACK (D-phenylalanyl-L-prolyl-arginyl chloromethyl ketone), he found that it completely repressed tau breakdown, whereas in the absence of the inhibitor, tau was degraded into short (To determine which peptide bonds are severed by the protease, Arai and colleagues incubated recombinant tau with purified thrombin and analyzed the resulting peptide fragments. This revealed that thrombin cuts tau in five locations (after Arg155, Arg209, Arg230, Lys340, and Lys257). The time course of proteolysis suggests that the Arg155-Gly156 bond is hydrolyzed first and that the remaining C-terminal end of tau is then cleaved from both the N- and C-terminal directions.

But what about phosphorylated tau, which is, after all, the most toxic form? Arai found that if tau is first phosphorylated with glycogen synthase kinase 3β (GSK-3β, which has been implicated in tau phosphorylation and may be a viable drug target; see ARF related news story), then the first thrombin cleavage, which normally takes place within seconds, proceeds much more slowly. For example, using Western blotting, Arai found that full-length unphosphorylated tau is completely eliminated after a three-hour incubation with thrombin, whereas about 50 percent of phosphorylated tau remains. Phosphorylation may also prevent the other four cleavages completely, because though smaller fragments of about 25, 23, and 16 KDa resulted from incubating unphosphorylated tau with thrombin, the authors failed to detect these peptides when they incubated phosphorylated tau with the protease. Physiologically, these observations may be relevant because when Arai used thrombin to digest paired-helical fragments of tau isolated from Alzheimer disease brain tissue, he found that digestion of the protein was negligible unless it was first incubated with alkaline phosphatase. This suggests that thrombin may have difficulty degrading excess phosphorylated tau in vivo, leaving it available for incorporation into neurofibrillary tangles.

Which brings us to the second paper from Cheng-Xin Gong and colleagues at the New York State Institute for Basic Research and Purdue University, Indiana. First author Fei Liu and colleagues report that protein phosphatase 5 (PP5) rivals protein phosphatase 2A (PP2A) in dephosphorylating tau.

In comparison to the hundreds of known kinases, there are only a handful of phosphatases and each of them must dephosphorylate a wide range of protein substrates. PP1, PP2A, and PP2B have all been shown to dephosphorylate tau in vitro, but PP2A may play a major role in vivo because in transgenic mice with reduced PP2A activity, tau becomes hyperphosphorylated and redistributed in CNS neurons (see Kins et al., 2001), while reduced expression of the phosphatase in the brain has been associated with AD (see, for example, Vogelsberg-Ragaglia et al., 2001). However, PP2A does not dephosphorylate all the sites on tau, suggesting that other phosphatases may be equally important in vivo.

When Liu and colleagues tested recombinant PP5 in a test tube assay, they found that it dephosphorylated tau about as well as PP2A does, but not necessarily at the same sites. There is evidence to suggest that PP2A dephosphorylates serines 262, 356, 396, and 404 most efficiently (see Sun et al., 2003), but Liu found that PP5 preferentially dephosphorylated tau at threonine 205, 212, and serine 409.

So are PP2A and PP5 cooperating to remove phosphate groups from tau in vivo? Perhaps. When Liu and colleagues measured PP5 activity in brain extracts from AD patients, they found it was reduced by about 20 percent. Expression levels were no different than in control samples, however, indicating that the enzymatic activity may be modulated in AD brain. The authors suggest that the phosphatase may be post-translationally modified or that an inhibitor may copurify with the enzyme to poison the phosphatase assay.

Indeed, protein partners are well known to modify protein phosphatase activities. Just recently, work from Bradley Denker and colleagues at Brigham and Women’s Hospital and Harvard Medical School showed that the G protein Gα12 interacts with the scaffolding subunit (Aα) of PP2A, enhancing its dephosphorylation of tau.

First author Deguang Zhu and colleagues found that Gα and Aα coimmunoprecipitate, and using immunofluorescence, the authors were also able to show that the proteins colocalize in primary neurons. In addition, the researchers found that adding Gα12 to PP2A in vitro elicited a threefold increase in phosphatase activity. The authors also found that, when expressed in COS cells that produce copious amounts of phosphorylated tau, Gα12 was found to stimulate dephosphorylation of tau by about 60 percent.

Ironically, another ligand of Gα12 is thrombin, suggesting that this G protein may regulate both the phosphatase and protease activities that are necessary to prevent accumulation of toxic tau.—Tom Fagan

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Of the diverse range of posttranslational modifications of tau (reviewed by us in Chen et al., 2004), serine/threonine-directed phosphorylation and truncations are among the most intensely studied, yet the role of their relative contribution to the tau pathology in Alzheimer disease is not entirely understood. Additional light is shed on them by four recent publications which now appear in JBC (in press). These publications deal with the dephosphorylation of tau by PP5 (Gong and coworkers), Gα12-stimulated PP2A activity and dephosphorylation of tau (Brad Denker and coworkers), thrombin-mediated cleavage of tau (Patrick McGeer and coworkers), and tau phosphorylation and cleavage during apoptosis (Faraj Terro and coworkers). A combination of studies with recombinant proteins, tissue culture experiments and the analysis of human brain section aims to dissect the role of these modifications (and the enzymes responsible for them) under both physiologic and pathologic conditions. As several enzymes seem able to phosphorylate or truncate tau (although to different degrees at different sites), questions remain. For example, how specific are these reactions and how do enzymatic activities overlap? As Gong and coworkers point out for activities of the two phosphatases PP2A and PP5 with respect to tau, "these studies suggest that tau phosphorylation can be regulated by more than one phosphatase, and that each phosphatase may regulate tau phosphorylation at different sites with a certain preference." This even points to the possibility that conditions may vary for different cell types and different metabolic conditions, a question not much addressed in the past. In addition, in the living organism, the situation may be different from those in tissue culture or cell-free extracts. When we used a dominant negative mutant approach to interfere with PP2A function, we found that only two epitopes, the AT8 and the pS422 epitope, were hyperphosphorylated, much different from the in vitro situation (Kins et al., 2001). We recently confirmed these findings with a new transgenic mouse strain. In addition, PP2A (and possibly also PP5) may not only act directly on tau, but also indirectly, by activating distinct kinases (Pei et al., 2003 and Kins et al., 2003).

The identification of Gα12 as a PP2A interactor is particularly interesting because Gα12 has been shown also to interact with cadherin, thereby affecting β-catenin signaling. PP2A itself is part of the wnt signaling complex composed of axin, APC, and GSK3. That the tau protease thrombin binds to Gα12 points to a complicated interplay of different cellular components that is not easy to dissect. While all of these findings should help us to understand tau pathology, one thing should be kept in mind: If tau aggregation is a seeding phenomenon, then the components that initially trigger tau aggregation (such as tau species of distinct size and a distinct phosphorylation pattern) may be underrepresented in any extract obtained from AD brains.